Thursday, December 31, 2015

Had a bit of a Tweet scream about people promoting fecal transplants to cure all sorts of ailments. Yes fecal transplants are amazing, for C. diff infections. And yes, they are worth testing for other ailments connected to microbes and inflammation. But worth testing is very different than "they should be used for X". So I posted a bit about this. Here is a Storify summary of some of the discussion

Thursday, December 17, 2015

Just had an interesting discussion on Twitter about the (what I see as silly) restrictions placed on giving names to bacterial and archaeal species where approved names are reserved for cultured organisms. Uncultured organisms have to use "Candidatus" (see Wikipedia here).

Sunday, December 13, 2015

I saw this post by Craig Pikaard on Facebook and it brought back some memories:

New paper from my lab in which we identified the RNAs made by RNA Polymerase IV, an enzyme we discovered ~15 years ago. Took us more than ten years to find the little buggers, but we finally got 'em. The paper is "open access", meaning that anyone can read it without paying a download fee or subscription. So have at it if you need a nap.

And the post included a link to a new paper in Elife. This brought back memories because I had a small part in the discovery (or more accurately, some post discovery analysis). So - let's step into a time machine here provided by, well, me keeping all my email forever I guess.

It was September 2000. I was working as a faculty member at TIGR (The Institute for Genomic Research) and I was doing some evolutionary analysis of the Arabidopsis thaliana genome, for what would become my most highly cited paper: Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. And then on Sept 6 day I got an email from someone who I had gotten to know a little bit who was also analyzing the genome:

----------------------------------
9/6

Dear Jonathan,

In helping Mike Bevan search for the general transcription machinery, I've
stumbled across something odd that might also interest you given its
evolutionary implications.

There should be three related genes in the Arabidopsis genome (or more if
any of the genes are duplicated) encoding ~135 kd (2nd largest)
DNA-dependent RNA polymerase subunits - one each for pol I, II and III.
These subunits are similar and are clearly related to one another (also to
the B subunit of the single bacterial RNA polymerase) yet they have
distinct motifs that allow them to be placed in each class (pol I, II, or
III) based on clustal analysis with orthologs from other species. Anyway,
there ARE three distinct ~135 kd subunit genes in the thaliana genome and
based on multiple alignments vs. mouse, yeast, drosophila etc genes, and
clustal analysis to draw phylogenetic trees, one is clearly for pol II, and
one is clearly for pol III. The third paralog is strange- it does not group
with other pol I 135 kd subunits (from yeast, Drosophila, Euplotes, mouse,
C. elegans), nor with pol II or III subunits. In fact, it appears as an
outgroup even when archael subunits (e.g. Sulfolobus) are included in the
analysis: archael subunits are more closely related to the pol II second
largest subunit than the mystery subunit is to other pol I, II, or III
subunits. By BLAST searching Genbank, the mystery subunit does not match
anything better than eukaryotic 135 kd subunits and it doesn't look like a
chloroplast or mitochondrial subunit. I'm wondering if a plant Pol I can
really be that weird.

Is this something you would be interested in looking at if I send you the
protein sequences for clustal analysis?

Now this certainly seemed interesting and as I was doing a variety of analyses of RNA polymerase homologs for some studies of the evolution of microbes, it was something I actually knew a little bit about. So I wrote back immediately:

Craig

This sounds quite interesting. I have found that for many of the DNA repair genes I have been looking at, the A. thaliana genes do show quite long branches, so long branches might be a possibility. A good phylogenetic analysis should be able to detemrine if that is the case. If you send me the sequences and/or an alignment, I would be happy to put them through a more deailed phylogenetic analysis.

Jonathan

Then, a few minutes later I got another email:

Hi Jonathan,

I'm pasting below the sequences I used for the multiple alignments (using
DNAStar), starting with the mystery gene and then known second subunits of
pol I, II, III, and archae.
Thanks for having a look at this.
Craig
-----------

largest subunitMEYNEYEPEPQYVEDDDDEEITQEDAWAVISAYFEEKGLVRQQLDSFDEFIQNTMQEIVDESADIEIRPESQHNPGHQSDFAETIYKISFGQIYLSKPMMTESDGETATLFPKAARLRNLTYSAPLYVDVTKRVIKKGHDGEEVTETQDFTKVFIGKVPIMLRSSYCTLFQNSEKDLTELGECPYDQGGYFIINGSEKVLIAQEKMSTNHVYVFKKRQPNKYAYVGEVRSMAENQNRPPSTMFVRMLARASAKGGSSGQYIRCTLPYIRTEIPIIIVFRALGFVADKDILEHICYDFADTQMMELLRPSLEEAFVIQNQLVALDYIGKRGATVGVTKEKRIKYARDILQKEMLPHVGIGEHCETKKAYYFGYIIHRLLLCALGRRPEDDRDHYGNKRLDLAGPLLGGLFRMLFRKLTRDVRSYVQKCVDNGKEVNLQFAIKAKTITSGLKYSLATGNWGQANAAGTRAGVSQVLNRLTYASTLSHLRRLNSPIGREGKLAKPRQLHNSQWGMMCPAETPEGQACGLVKNLALMVYITVGSAAYPILEFLEEWGTENFEEISPSVIPQATKIFVNGMWVGVHRDPDMLVKTLRRLRRRVDVNTEVGVVRDIRLKELRIYTDYGRCSRPLFIVDNQKLLIKKRDIYALQQRESAEEDGWHHLVAKGFIEYIDTEEEETTMISMTISDLVQARLRPEEAYTENYTHCEIHPSLILGVCASIIPFPDHNQSPRNTYQSAMGKQAMGIYVTNYQFRMDTLAYVLYYPQKPLVTTRAMEHLHFRQLPAGINAIVAISCYSGYNQEDSVIMNQSSIDRGFFRSLFFRSYRDEEKKMGTLVKEDFGRPDRGSTMGMRHGSYDKLDDDGLAPPGTRVSGEDVIIGKTTPISQDEAQGQSSRYTRRDHSISLRHSETGMVDQVLLTTNADGLRFVKVRVRSVRIPQIGDKFSSRHGQKGTVGMTYTQEDMPWTIEGVTPDIIVNPHAIPSRMTIGQLIECIMGKVAAHMGKEGDATPFTDVTVDNISKALHKCGYQMRGFERMYNGHTGRPLTAMIFLGPTYYQRLKHMVDDKIHSRGRGPVQILTRQPAEGRSRDGGLRFGEMERDCMIAHGAAHFLKERLFDQSDAYRVHVCEVCGLIAIANLKKNSFECRGCKNKTDIVQVYIPYACKLLFQELMSMAIAPRMLTKHLKSAKGRQ-----------------

Well, this was helpful. Sequences and useful notes about them. So I played around with the sequences and searched for some other homologs and built a few alignments, build some masks to filter out poorly aligned regions, and then fed the data into PAUP and built a tree. (I note - I know about this because amazingly I still have all the files)

And I wrote back to Mike Bevan and Craig on Sept 8:

Mike and Craig

Attached is a phylogenetic tree of RNA polymerase subunits (Craig suggested I look at these because of an unusual protein in the A. thaliana genome). A. thaliana has representatives in five different subfamilies - Pol-I, Pol-II, Pol-III and RpoB (for the chloroplast) as would be expected and then this novel Pol which I have called Pol-IV.

I do not know much about RNA polymerase, but it seems like this is a pretty big deal and I think should be emphasized in the paper. What do you think? I could try to make a pretty tree figure to show the different families.

Jonathan

I got an email back:

Dear Jonathan (and Mike),

Many thanks for the detailed phylogenetic tree of the mystery pol subunit.
I think a figure is the only way to show clearly that this protein defines
a new clade. Is there room for such a figure, Mike?

In the lab we have also been calling it a putative pol IV subunit just for
the shock value of saying the words (a radical idea in the transcription
field), though in the absence of knowing what other subunits associate with
it, I'm not sure what to call it in the annotation or figure. Maybe
"oddpol" or "atypical polymerase 2nd subunit". It takes more than a dozen
subunits to make a eukaryotic polymerase, so it is not clear that one
unusual subunit is enough to confer new properties-i.e. a true pol IV.
Obviously, that will require quite a bit of work.

Cheers,
Craig

Me to Craig on 9/11/00:

Yes

I agree that it is too early to call it a true polIV, and I was doing it for the shock value too

Jonathan

PS. Do you mind if I present this at the TIGR GSAC meeting later this week

Jonathan

Craig to me 9/11:

Hi Jonathan,

Feel free to show the data. In thinking more about this, it is worth also
making a phylogenetic tree for the largest pol subunit (the equivalent of
eubacterial B') just to see if there might be a fourth class out there for
the largest subunit, too. If there is, pol IV may not be such a wild idea.

In case you are interested in giving this a try, I'm including some
sequences below. In the meantime, is there a good web site for performing
the types of extensive phylogenetic trees you've done for the mystery
subunit? I should do this for many of the general transcription factors
just to be sure they really group with the correct homologs, as you
suggested.

Anyway, here are some largest subunit sequences for pol I, II and III.
Vive la difference!

sorry .. no useful sites out there for doing phylogenetic analysis ... I am working on such a type of thing right now. I tis tricky becuase to do it correctly you need to filter out parts of a multiple sequence alignment to remove badly aligned regions as well as hypervariable regions.

9/12 Craig to Me

Dear Mike,

Yes, I can do this for the atypical RNA polymerase 2nd subunit. I have
already done multiple alignments with it against pol I, II, III subunits
and it is clear that the atypical subunit has amino acid differences that
set it apart, rather than large indels that skew the data. So I think
Jonathan is safe to go ahead and make a figure while I examine the gene
sequences and gene models more carefully.

Any comments on the tone/amount of detail in the section I wrote on the
general transcription machinery? Either way, I will add some references
and send you an updated version as soon as I can.

cheers
Craig

---------------------
>Speaking on behalf of the editorial committee whom I have not consulted, I
>would be delighted to have this in our section. But we need to check out the
>gene structure in detail (dodgy gene prediction, missing exons etc. Craig,
>could you so this as you know most about these enzymes
>
>All the best
>
>Mike

Me to Craig

Craig

I am still working on a slightly better figure ... but I have attached the latest version ... I think it is sufficient for submission

I have attached it in a few different formats.

I will be out of town for a few days but checking email.

Jonathan

Craig to Me:

Hi Jonathan,

The phytlogenetic tree figure for the atypical pol subunit looks good
though the font size may need to be reduced to fit "Fungal Plasmids"
between the dividing lines for the adjacent categories. Have you sent a
copy to Mike?

Craig

Craig again

Hi Jonathan,

I forwarded a copy to Mike. Did you ever have a chance to do a tree for the
largest subunit to further test the hypothesis of a pol IV?

Hope you are having fun in LA

Craig

> I am not sure if I sent a copy to mike
>
>I am in LA right now and it would be easier if you could send mike a copy to
>make sure he has one. I will try and edit the figure and send one with a
>smaller font.
>
>J

10/3 Me to Craig:

Criag

Attached is a new version of the rna pol tree with fonts corrected. I am going to add a few more sequences a rerun it and make a new tree tomorrow.

Jonathan

PS Also ... here is a potential figure legend

Figure. Phylogenetic tree of RNA polymerase homologs. Homologs of RNA polymerase were identified by searching sequence databases with representatives of the major known RNA polymerase subfamilies. These proteins, as well as six DNA polymerase homologs from A. thaliana, were aligned using clustalx using default settings. Phylogenetic trees were generated from the alignment (with ambiguously aligned regions and hypervariable regions excluded) using the PAUP* program. The tree shows was generated using the neighbor-joining algorithm with pairwise distances between sequences calculated with a PAM-like matrix. Numbers on the branches are bootstrap values indicating the percentage of 100 trees in which the proteins to the right of the node grouped together to the exclusion of all other proteins.

Craig 10/3

Hi Jonathan,

I will look forward to seeing the final tree, as will Mike, I'm sure. For
the legend, the fact that this is an alignment of second-largest subunits
should be made clear. Here is a stab at a minor revision:

Figure-----. Phylogenetic tree for the second-largest subunit of
DNA-dependent RNA polymerases. Homologs of RNA polymerase second-largest
subunits were identified by searching sequence databases with
representatives of the major known subfamilies (e.g. pol I, II, III and
eubacterial beta subunits). Identified proteins, including six homologs
from A. thaliana, were
aligned using clustalx using default settings. Phylogenetic trees were
generated from the alignment (with ambiguously aligned regions and
hypervariable regions excluded) using the PAUP* program. The treewas generated using the neighbor-joining algorithm with pairwise distances
between sequences calculated with a PAM-like matrix. Numbers on the
branches are bootstrap values indicating the percentage of 100 trees inwhich the proteins to the right of the node group together to the
exclusion of all other proteins.

Thanks,
Craig

Me:

much better figure legend

j

Anyway - and so it went. Alas, for a variety of reasons not much made it into the final paper. What was there was this:

Unexpectedly, Arabidopsis has two genes encoding a fourth class of largest subunit and second-largest subunit (Supplementary Information Fig. 5). It will be interesting to determine whether the atypical subunits comprise a polymerase that has a plant-specific function.

And of course, this Supplemental Information is not exactly easy to find and does not actually work correctly anymore:

Downloading the Zip file and opening first page.htm gets one to this

And then clicking on the Figure 5 you get a broken page w/o the Figure.

But there, hidden in the folder with the Supplemental Information is the figure

So that is the beginning of the story on RNA Pol IV in Arabidopsis.

Go read the E-life paper and some of what it cites for the last 15 years of the story.

I am going to skip over the main Box 1 for now. And go to Box 2 - Terminology because this will be important for the rest of the article

Box 2. Terminology

Coevolution: reciprocal evolution of interacting species

Seems OK.

Commensalism: a relationship benefiting one party while the other is unaffected

I am good with this. Though I do wonder if they mean that this is a form of symbiosis or if it applies to any interaction even one that is not a symbiosis. When I teach this I treat commensalism as a form of symbiosis.

Mutualism: a relationship benefiting both parties

Same comment as for commensalism

Parasitism: a relationship benefiting one party to the other’s detriment

Same comment as for commensalism and mutualism.

Symbiosis: two or more species living closely together in a long-term relationship

I am good with this

Macrobe: a eukaryotic host, most being visible by eye

I don't think I like this. Why does a macrobe have to be a eukaryote?

Microbiota: the microbes in or on a host, including bacteria, archaea, viruses, protists, and fungi

Holobiont: a unit of biological organization composed of a host and its microbiota

I am OK with this if the authors what to use it as a proposed definition to then test. However, I am not at this point convinced that a there is a "unit" of biological organization here. I would I guess prefer a holobiont to be defined as a "host and its microbiota" without the "unit of biological organization part"

Hologenome: the complete genetic content of the host genome, its organelles’ genomes, and its microbiome

I am OK with this as a proposed definition.

Microbe flow: the exchange of microbes between holobionts

Seems OK.

Phylosymbiosis: microbial community relationships changing in parallel with the host nuclear phylogeny

This seems potentially very useful to distinguish something from co-evolution.

Hologenome Concept of EvolutionThe hologenome concept of evolution was first explicitly introduced in 1994 during a symposium lecture by Richard Jefferson [56], and it was independently derived in 2007 by Eugene Rosenberg and Ilana Zilber-Rosenberg [57].

Good to see some of the history here. And since Richard Jefferson has been talking to me about this for many years it is good to see him mentioned.

It posits that hosts and their microbiota are emergent individuals, or holobionts, that exhibit synergistic phenotypes that are subject to evolutionary forces [35–37].

I am fine with this as a "posit". Not saying I agree it is true. But here it is presented clearly as what the Hologenome Concept implies.

Via fidelity of transmission from parents to offspring or stable acquisition of the microbiome from the environment, covariance between the host and microbiota can be established and maintained.

OK again as an explanation of the model.

Consequently, as with phenotypes encoded by nuclear genomes, phenotypes encoded by beneficial, deleterious, and neutral microbes in the microbiome are subject to selection and drift within holobiont populations. Genetic variation among hologenomes can arise through changes to host genomes as well as through changes to the genomes of constituent symbiotic microbes [35–37,58]. The microbiomes, and thus their encoded phenotypes, can change through differences in the relative abundances of specific symbiotic microbes, the modification of the genomes of existing resident microbes, or the incorporation of new microbial symbionts into holobionts, which can occur even within the reproductive lifetime of hosts [58]. Importantly, genetic variation in the microbiome vastly exceeds that in the host genome and accumulates much more rapidly than variation in host genomes. Therefore, given that genetic variation is the raw material upon which evolution ultimately acts, microbial sources of hologenomic variation are potential targets of evolution, and, despite its inherent complexity, biologists must consider the incorporation of the microbiome in the overall study of evolution.

I am fine with all of this as an explanation of the model / concept. Again, not saying I think it is right but good to see it laid out pretty clearly.

Wednesday, December 09, 2015

There are many discussions going on about a paper from Bordenstein and Theis that was published in PLOS Biology in August 2015. The paper is Bordenstein SR, Theis KR (2015) Host Biology in Light of the Microbiome: Ten Principles of Holobionts and Hologenomes. PLoS Biol 13(8): e1002226. doi:10.1371/journal.pbio.1002226

I made some comments on Twitter when the 1st paper came out about how I was skeptical of the paper and in discussions with Seth Bordenstein I said I would try to write up my thoughts. And when I was pointed to the second paper today I posted to Twitter that I thought it was important and got into a brief discussion with Seth about the paper.

In thinking about the papers and science publishing and scientific discussions I have decicded to try and carry out a new experiment. I am going to go, as fast as I can, line for line through the papers and post my thoughts in response to those lines. And I will try to be honest even if my thoughts are not, well thought out or nice or helpful. I am just going to post the thoughts. And one reason I want to do this is I worry (or maybe realize) that my judgement may be being affected here by visceral responses to some of the lines. In particular, I confess, some of the way the Bordenstein and Theis article is written really rubs me the wrong way. Nothing personal against the authors. But the text did not agree with me in parts. And I think that may have affected my response to the article. I do not know for sure but it seems possible.

Regardless, I am going to try and go through this. And for now I am going to just start with the Abstract.

Groundbreaking research on the universality and diversity of microorganisms is now challenging the life sciences to upgrade fundamental theories that once seemed untouchable.

I personally find this to be a bit too extreme. Really - did they once seem untouchable? To whom?

To fully appreciate the change that the field is now undergoing, one has to place the epochs and foundational principles of Darwin, Mendel, and the modern synthesis in light of the current advances that are enabling a new vision for the central importance of microbiology.

I think it is overstating the "central importance of microbiology" to place it somehow in line with Darwin, Mendel and the modern synthesis

Animals and plants are no longer heralded as autonomous entities but rather as biomolecular networks composed of the host plus its associated microbes, i.e., "holobionts."

While on the one hand I agree with part of this statement I think it is making a claim and stating it as a fact when this is what is being debated.

As such, their collective genomes forge a "hologenome," and models of animal and plant biology that do not account for these intergenomic associations are incomplete.

Certainly animal and plant biology has to account for microbes. But it is false logic to say that one can only account for microbes by following the hologenome concepts.

Here, we integrate these concepts into historical and contemporary visions of biology and summarize a predictive and refutable framework for their evaluation.

No thoughts on this.

Specifically, we present ten principles that clarify and append what these concepts are and are not, explain how they both support and extend existing theory in the life sciences, and discuss their potential ramifications for the multifaceted approaches of zoology and botany.

Confession. Saying ones own principles "clarify" something rubs me the wrong way. I would really have preferred it if they said "attempt to clarify".

We anticipate that the conceptual and evidence-based foundation provided in this essay will serve as a roadmap for hypothesis-driven, experimentally validated research on holobionts and their hologenomes, thereby catalyzing the continued fusion of biology's subdisciplines.

I find this to be really overstated too. I don't think what you have presented in this paper is a roadmap. And for you to call it that sets up this essay as basically saying that everything else that has come before is limited and lame.

At a time when symbiotic microbes are recognized as fundamental to all aspects of animal and plant biology, the holobiont and hologenome concepts afford a holistic view of biological complexity that is consistent with the generally reductionist approaches of biology.

I do not think symbiotic microbes are fundamental to all aspects of animal and plant biology. I think this is actually a silly statement and makes me doubt the objectivity of the authors.

Based on your previous experience &amp; publications, we would like to invite you to join the editorial board of the journal “Austin Biology”.

About the journal

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Website Links

Journal home page: http://austinpublishinggroup.com/biology/

Our aim is to publish the latest information &amp; provide a platform for all Biology for mutual exchange of ideas, thoughts.

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